Mitochondrial ATP-dependent proteases are vital for maintaining cellular homeostasis and the response to environmental stress. These enzymes are ATP-powered proteolytic machines that selectively degrade abnormal proteins and regulate metabolic processes. In many disease states and aging, the increased generation of reactive oxygen species within mitochondria results in protein oxidation and aggregation. As a counter-measure, the mitochondrial Lon protease for example, degrades oxidized aconitase thereby preventing its irreversible accumulation and aggregation. The oxidative damage of proteins, nucleic acids and lipids is directly linked to aging, heart disease and neuromuscular disorders;whereas protein aggregation is common to many neurodegenerative disorders. In addition, recent work suggests that Lon-mediated proteolysis may be important in tumorigenesis and the adaptation of intratumoral cells to hypoxia. Unfortunately, there are no specific high affinity inhibitors or activators of mitochondrial ATP-dependent proteases. The goals of this proposal are to develop assays for identifying and validating compounds that selectively activate or inhibit the Lon protease. Aim 1 is to develop and optimize a primary screening assay for measuring Lonmediated degradation of reporter peptide substrate using time-resolved fluorometry. The feasibility and reproducibility of these assays will be demonstrated using commercially available chemical libraries. Aim 2 is to develop and optimize secondary- and counter- screening assays, which will provide information about the mechanism of compound activity, and distinguish compounds that are Lon-specific from those that target other mitochondrial or non-mitochondrial proteases. Small molecule activators of mitochondrial ATP-dependent proteolysis have potential application in the treatment of neurodegenerative and/or myocardial dysfunctions linked to mitochondrial protein aggregation. Our preliminary data suggest that inhibitors of mitochondrial ATP-dependent proteolysis may function as anti-cancer agents, with potential clinical application either alone or in combination with other chemotherapeutic strategies. Taken together, Lon and other mitochondrial ATP-dependent proteases may be new and viable drug targets.